定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

Effect of site-directed mutagenesis on soluble expression and specific activity of amide hydrolase DamH

摘要:

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶，其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成，并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变，通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用，通过Ni2+- NTA亲和层析对D165及N192突变子进行纯化，对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%，E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达，表达量分别为野生型的28.2%和20.8%，突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274，其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。

Abstract:

Abstract:［Objective］ DamH is a bifunctional hydrolase that hydrolyzes the amide and ester bonds．Previous studies demonstrated that mutagenesis of non-catalytic residues shows a negative impact on the soluble expression and specific activity of DamH．Therefore，we studied the catalytic triad of DamH and the effect of non-catalytic mutagenesis on the soluble expression and specific activity of DamH.［Methods］We performed site-directed mutation experiment of the 3 possible catalytic sites: S149，E244 and H274 and the non-active sites by using overlapping PCR．In the whole cell catalytic experiments，2 ’-methyl-6 ’-ethyl-2-chloroacetanilide ( CMEPA ) hydrolase activities of the three mutants (S149A，E244A and H274A) were assayed．Mutants of D165 and N192 were purified by affinity chromatography of Ni2+- NTA．At the same time the hydrolase activities of mutants were compared with that of the wild-type strain．［Results］S149-E244-H274 was the catalytic triad of DamH． Kinetics shows that the CMEPA hydrolase activity of mutant S149A declined to 5% of the wild-type strain and none of E244A and H274A mutants showed any CMEPA hydrolase activity．Mutations of D165 and N192 would affect the soluble expression of recombinant enzyme． The soluble expression levels of D165P and N192P were 28. 2% and 20. 8% of the wild-type strain，respectively． Furthermore，hydrolase activities of N192P and D165P were only 55. 5% and 49. 7% of the wild-type enzyme，respectively．［Conclusion］DamH uses the same active site to hydrolyze esters and amides.